Recently, we discovered serendipitously that certain circular minidefensins, antimicrobial peptides structurally related to the theta-defensins recently described in rhesus macaques, potently inhibit HIV-1 infection in vitro. Although humans no longer produce circular minidefensins, human bone marrow provides evidence - in the form of an expressed pseudogene - that these peptides were once part of our genomic heritage. Using the sequence information still encoded within its expressed pseudogene, we recreated this lost human peptide which we have named retrocyclin. Retrocyclin proved to be a more effective antiretroviral than its rhesus counterparts, potently preventing infection of CD4+ cells by both T-and M-tropic HIV-1. Because it is a small (18 residue) and well-defined molecule, retrocyclin is a promising lead compound for designing topical agents that can be used to prevent human HIV-1 infections. Based on our preliminary studies, we hypothesize that retrocyclin a) acts by inhibiting viral attachment, fusion or entry; b) will inhibit HIV-1 infection in the mucosa and be active in mucosal fluids, and c) is a leading candidate for development as a topical vaginal or rectal microbicide to prevent HIV transmission. With assistance from a multidisciplinary team of immunologists, retrovirologists, and a peptide chemist, we will test these hypotheses by 1) characterizing determinants of retrocyclin activity and identifying the molecular target(s) for its potent inhibitory effects on HIV-1 infection, 2) characterizing structural determinants of retrocyclin activity through the rational design and testing of retrocyclin congeners, and 3) determining the stability of retrocyclin and congeners in human fluids. Such findings could elucidate novel mechanisms of natural resistance to HIV-1 and promote the development of novel, host-compatible antiretrovirals to prevent HIV infection. Because progress in developing an effective vaccine for HIV-1 has been slow, there is an urgent need to find alternative preventive approaches. Our long term goal is to develop the basic information needed to develop topical agents that prevent mucosal (particularly vaginal or rectal) transmission of HIV-1. Availability of such agents, in the form of retrocyclin-containing creams or gels, would empower vulnerable sexual partners by providing them with an invisible, effective means of protection.